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Ternary chromium(III)-nucleotide-amino acid complexes: L -methionine, L -serine and glycine derivatives
Inorganica Chimica Acta, 169
(1990) 133-139
133
Ternary Chromium(III)-Nucleotide-Amino L-Serine and Glycine Derivatives
Acid Complexes:
L-Methionine,
A. M. CALAFAT, J. J. FIOL, A. TERRON Departament de Quimica, Universitat de les Illes Balears, 07071 Palma de Mallorca (Spain)
V. MORENO Facultad de Quimica, Universitat de Barcelona, 43005
Tarragona (Spain)
D. M. L. GOODGAMEand I. HUSSAIN Department
of Chemistry, Imperial College, London SW72AY
(U.K.)
(Received May 22,1989; revised September 22,1989)
Abstract The first ternary Cr(III)-nucleotide-amino acid complexes (nucleotide: S’AMP, 5’CMP; amino acid: L-serine, L-methionine, glydne) are described. The complexes have been characterized by elemental and thermogravimetric analyses, IR and electronic spectroscopy and EPR measurements. In all cases the interaction of Cr(II1) with the nucleotide seems to occur mainly through the phosphate group, whereas the amino acid binds to Cr(II1) through the carboxylic and amino groups. Some of the complexes show distortions from octahedral geometry, but these distortions appear to be small resulting in values of the zero field splitting parameter D < 0.1 cm-‘. Introduction Although the role of chromium in biological systems is not perfectly known, there is abundant evidence that Cr(II1) is present in the glucose tolerance factor (GTF) [I, 21. For this reason, interest in derivatives of Cr(II1) with amino acids has increased during recent years [2-51. There is also great interest in obtaining inert derivatives of Cr(II1) with nucleotides, which could be used as enzymatic labels of allosteric enzymes [6] and for the study of the role of chromium in transcription processes and RNA and DNA interactions [7]. In this paper we describe the syntheses and characterization of the first ternary complexes of Cr(II1) with L-methionine, L-serine and glycine and the nucleotides 5’AMP and 5’CMP. Experimental Carbon, hydrogen, nitrogen and sulphur analyses were carried out with a Carlo Erba model 1106 0020-l 693/90/$3
SO
microanalyzer at the Centro de Investigation y Desarrollo-C.S.I.C. in Barcelona. Chlorine was determined by the Schoniger method. Chromium was determined by atomic absorption with a Perkin-Elmer 703 spectrophotometer using [Cr(urea)6]C1s*3Hz0 solutions as a standard. The phosphorus content was determined using the calorimetric method with phosphomolybdovanadate [8]. Thermogravimetric analyses were carried out with a Perkin-Elmer TGS-2 system with oxygen atmosphere at a velocity rate of 5 or 10 “/min depending on the cases. Conductivities were measured with a Crison 525 conductimeter at 25 “C in lop3 M aqueous solution. The IR spectra were recorded in solid state (KBr pellets) on a PerkinElmer 683 IR spectrophotometer connected to a Perkin-Elmer 3600 data station. The reflectance spectra were obtained with a Perkin-Elmer W-Vis spectrophotometer with an integrating sphere attachment. UV-Vis spectra were recorded in the same The EPR apparatus at 10e3- 1O-4 M concentration. spectra were measured using polycrystalline samples at room temperature. X-band spectra were obtained with a Varian E 12 spectrometer and Q-band spectra with a Bruker ER 200 D-SRC spectrometer and an ER 078 15-inch electromagnet (Imperial College, London). The nucleotides and amino acids (Fig. 1) were from Serva and Merck and used without further purification. The starting complex [Cr(urea)e] C13. 3Hz0 was prepared according to the literature [9]. Preparation Cr(_wnet)(L-metH)C12*3H20, Cr(L-ser)2(H20)2Cl. Hz0 and Cr(glyH)2(gly)C12*3H20 A 10 ml water solution containing 1 mmol of [Cr(urea)6]C13*3Hz0 (pH adjusted to 6.3) was added to a solution of amino acid (2 mmol in the case of L-methionine and L-serine and 3 mmol for glycine) 0 Elsevier Sequoia/Printed
in Switzerland
134
H,N-GH,-hOOH
(Gly)
GLYC INE
NH2
L-SERINE
HO-CH2-CH-COOH 2 1 3
( L-Ser)
L-METHIONINE(L-Met)
H3~-S-~H2-~H2~~H~~OOH
AH2 1;I”2
NH2
OH OH
OH OH 5’ AMP
5’ CMP
Fig. 1. Nucleotide used.
and amino
acid formulae
precipitate was formed. It was filtered off, washed with cold water and vacuum dried over P40i0. Cr2(L-ser)(5’AMPJ2CI.16H20 and Cr2(L-ser),(S’CMP)OHCl* 7H20 To an aqueous solution of Cr(L-ser)z(Hz0)2C1. Hz0 (0.94 mmol in 10 ml) was added dropwise a solution of Na25’XMP (0.94 mmol in 10 ml water, pH = 7). The violet resultant solution (pH = 4.54.7) was placed in a thermostated bath at 50 “C with constant stirring for 20 h (for the S’AMP complex) or 8 h (in the case of the S’CMP derivative). The final violet solution (pH = 3.4-3.6) was concentrated to 5 ml and then eluted through a Sephadex G-10 column to give two fractions: the first grey-blue, and the second violet. Addition of ethanol to the blue fraction and further evaporation gave and Cr*(L-ser),Cr’L(L-ser)(5’AMP),C1* 16Hz0 (S’CMP)(OH)Cl~7H,O, which are insoluble in water. These precipitates were vacuum dried over P40,e.
and abbreviations
in 10 ml water [pH adjusted to 9.2 (met and ser) and 9.8 (gly)]. The resultant green solution [pH = 9.2 (met), 9.3 (ser) and 9.7 (gly)] was placed in a thermostated bath at 50 “C, with constant stirring, for 10 h (5 h in the case of methionine complex), by which time a lilac-violet solution [pH = 3.7 (met), 3.3 (ser) and 3.9 (gly)] was obtained. This was concentrated in a rota-vapor and then eluted through a Sephadex G-10 column to give two fractions: the first one, violet, corresponding to the binary complex, and the second one, green, corresponding to unreacted starting complex. In the case of the glycine complex, there were three fractions: the first one, blue, in a very small quantity; the second, violet, corresponding to the binary complex; the third, green, corresponding to unreacted starting complex. A precipitate corresponding to the violet fraction was obtained on evaporating the solution or adding ethanol (for the L-serine complex). This was vacuum dried over P,O,,,. The three complexes are hygroscopic and soluble in water. Cr(L-ser)2(L-serH)Cl*3H20 This was obtained by the same procedure as Cr(L-ser)2(H20)2C1*H~0, but using a 1:3 Cr:L-ser stoichiometry. Cr(L-met)(5’AMP)*$H20 and Crz(L-met)(5’CMP)20H~10Hz0 Ten ml of an aqueous solution (pH approx. 7) containing 0.75 mmol of Na25’XMP were added dropwise to a solution of Cr(L-met)(L-metH)&. 3HaO (0.75 mmol in 10 ml HzO, pH 3.9-4.2). The violet mixture (pH 5.6-5.7) was maintained at 50 “C, with constant stirring, for 1.5-2.5 h. A grey
Cr2(gly)2(5’AMP)(OH)2*6H20 and Cr2(gly)2(5’CMP)(OH),~6H,0 Ten ml of an aqueous solution containing 0.64 mmol Na25’XMP (pH adjusted to 7.0) was added dropwise to an aqueous solution of Cr(gly)(glyH),C12*3Hz0 (0.64 mmol in 10 ml). The violet solution (pH 5.5 or 6.1) was maintained at 50 “C with continuous stirring for 5 or 8 h, depending on the nucleotide. By this time a precipitate was formed. This was filtered off, washed with cold water and then vacuum dried over P40io. The composition of the complexes and the analytical results are reported in Table 1. The elemental analyses were confirmed by the thermogravimetric studies, which are summarized in Table 2.
Results and Discussion The Cr(III)-amino acid binary complexes are very hygroscopic and soluble in water. Their molar conductivities are consistent with non-coordination of the chlorine atoms to chromium [lo]. The fact that the molar conductivity value of Cr(L-met)(L-metH)C12*3H20 is greater than expected for an electrolyte 1:2 may be explained as a result of dissociation equilibrium from the amino acid. The infrared spectra of these complexes show in all cases, modifications of the bands related to vibrations of the carboxylic group suggesting that this group is involved in the coordination to Cr(III) [ II131. The most important changes occur in the v,(COO-) bands (1412, 1420 and 1416 cm-i for L-met, L-ser and gly respectively) which are shifted 20-30 cm-’ to lower frequencies. There are also noticeable modifications in the (500-600) cm’
135
0
N
136 TABLE
2. Thermogravimetric
Compound
Cr(L-met)(L-metH)Cla*3HaO
Cr(L-met)(S’AMP)*11/2HaO
data for the complexes
Temperature
(“C)
30-l 30 130-980 residue 30-120
Tentative
Weight loss (%)
assignment
Calc.
Found
3.19 85.46
3.82 86.65
9.78
10.28
;HaO
68.31
2HaO + met + ado chromium phosphate
(esp.)
(esp.)
Hz0 2HaO + 2Cl+ 2met chromium oxide
120-980 residue
70.21
30-l 10 110-980 residue
8.24 66.43
8.33 66.54
5HaO met + 2cyd + 5HaO chromium phosphate
Cra(L-serH)a(L-ser)4C12.6H20
30-l 10 110-380 380-670 residue
5.94 73.28 9.18
6.25 73.26 9.10
3HaO 6ser + 2HaO Ha0 + 2Cl chromium oxide
Cr(L-ser)a(HaO)aCl*HaO
30-90 go-550 residue
5.15 80.55
5.21 79.23
Ha0 2H20 + 2ser + Cl chromium oxide
30-l 20 120-980 residue
6.65 69.05
6.67 71.13
30-l 10 110-980 residue
7.36 71.44
1.29 71.72
5HaO 1 lHa0 + Cl + 2ser + ado chromium phosphate (esp.)
30-600 residue
87.27
85.57
31~20 + 3gly + 2Cl chromium oxide
30-l 10 110-980 residue
9.74 61.32
9.12 60.80
4H20 2HaO + 2gly + ado chromium phosphate
(esp.)
30-l 20 120-980 residue
7.36 63.56
3HaO 4HaO + 2gly + cyd chromium phosphate
(esp.)
Cra(L-ser)(S’AMP)aCI.l6HaO
bands due to S(COO-), r,(COO-) and r,(COO-). The modifications of the 6,(NH2) bands at 15051514 cm-’ may be due to the interaction with Cr(II1) or to the participation of this group in hydrogen bonding [12]. The small variations in the bands arising from vibrations n(OH) at 805 cm-’ and V(S-CHs) at 13 19 cm-’ do not necessarily imply coordination of these groups to Cr(III). For Cr(L-met)(L-metH)C1,.3H,O, the bands at 578 and 450 cm-’ have been tentatively assigned to v(Cr-0) and y(Cr-N) respectively. There is no evidence of the presence of y(Cr-Cl) bands, in agreement with the conductivity measurements. Table 3 records the electronic spectral data which are in agreement with coordination of chromium to N and 0 donors in a pseudooctahedral geometry. These results are confirmed by EPR measurements. The X-band EPR spectra of the complexes Cr(L-met)(L-metH)Clz*3Hz0 and Cr(L-ser),(H,O),Cl.H,O,
4H20 + 2ser + cyd + Cl chromium phosphate (esp.)
7.22 62.76
gave a broad featureless band in the g,, = 2 region, characteristic of Cr(II1) in a pseudooctahedral environment. However, Cr(glyH)2(gly)Clz*3Hz0 and Cr(L-ser),(L-serH)Cl*3Hz0 gave EPR spectra comprising several overlapping bands in the range 0.150.4 T (Fig. 2). Such spectra may be described by the spin Hamiltonian for S = $ systems of the form ;fc = Pk.&S,
+&J&S,
+gJL%)
+ D(S,2 - 5/4) t E&2
- S,‘)
in which the second and third terms are the axial and rhombic zero field splittings (zfs) respectively [ 141. Although the individual band components are insufficiently resolved for accurate evaluation of D and E parameters, the facts that the strongest transition appears in the geff = 2 region, the bands below 0.2 T are very weak, and no bands are observed between 0.5-l T indicate a relatively low value for
137 TABLE
3. Electronic
spectral
data for the complexes
Compound
(bands
in nm)*
A-n* n-+n*
W4T
r,(P)
- 4A2a
(v J4Tza
+ 4A*g
10 Dq (cm-‘)
Cr(L-met)(L;metH)Cla.3H20
404(89)
548(41)
18200
Cr(L-ser)a(H20)2C1.H20
403(110)
541(60)
18500
Cra(L-serH)a(L-ser)4Cla.6H20
401(152)
541(105)
18500
403(84)
544(51)
18400
435s 395s
610s 570s 470sh
18200
440s 395s
610s 565s 500sh 465sh
18700
435s 395s
610s 570s 470sh
18200
27Os, 310s
435s 395s
610s 570s
17000
280sh,
435s 395s 365sh
615s 560s SOOsh 465sh
18700
435s 400s
610s 575s 500sh 470sh
18600
Cr(glyH)a(gly)C12.3Ha0 Naa5’AMP
b
252s, 293s
Cr(L-met)(5’AMP).11/2H20
b
Cra(L-ser)(S’AMP)2C1.16H20
b
Cr&ly)a(5’AMP)(OH)a.6H20
Naa5’CMP
255s
% = strong,
270br,
b
m = medium,
325s
255s, 290s
b
26Os, 308s
Cra(L-met)(5’CMP)2(0H).10H20
Cra(gly)a(5’CMP)(OH)2.7H20
295s
b
b
br = broad,
270s
325s
310s
sh = shoulder,
sp = sharp, w = weak.
D (
bDiffuse
reflectance
spectra.
ligand are not observed. However, this fact does not necessarily imply coordination through sulphur atom. In both complexes, the v,(COO-) band is shifted by 20-30 cm-’ to lower frequencies. The bands assignable to amino group vibrations are modified in the spectra of the complexes or overlap with nucleotide bands. With regard to nucleotide vibrations, there are small variations in some Y(ring) bands (1600-1200 cm-‘). In the case of the S’CMP compound, a new band appears at 1720 cm-’ which may be due to a free carboxylic group or to participation of C=O in hydrogen bonding upon complexation [18, 191. Nevertheless, the most important changes occur in the phosphate bands, especially that of u,(POa) band (978 cm-‘), which is shifted by 20-25 cm-’ to higher frequencies in both cases. Therefore, IR data suggest that methionine interacts with chromium through its carboxylic and amino groups [ll, 121 and the phosphate group of S’AMP and S’CMP is involved in metal coordination [ 18,201. In the case of serine ternary compounds again there are important changes in the bands correspond-
138
2
1
L
3
5
6 10’
mT
Fig. 2. X-band EPR spectra of polycrystalline samples of: (a) Cr+ser)(S’AMP)?Cl.l6HzO; (b) Crz(L-serH)2(L-ser)4C12*6Hz0; (c) Cr(glyH)2(gly)Clz*3HzO.
/
g=
(b)
.‘M,
9:
Cc)
C.-
/.‘. .-
.f ,
/:
1:
i! ;:
/// 9=
‘
6
.3
IO
12
102.7
Fig. 3. Q-band EPR spectra of pofycrystalline samples of: (a) (-. -) Crz(L-ser)(S’AMP)$l*16H20; (b) (-) CIZ(LserH)&ser)&12*6HzO; (c) (- - - -) Cr(glylI)~(gly)Cl~.3H~O.
ing to carboxylic vibrations. The band assignable to v,(COO-) shifts 25-30 cm-’ to lower frequencies, overlapping with -/,(CH,) + v(C-0) of the serine
(1384 cm-‘). The carboxylic bands at lower frequency, S(COO-) at 612 cm-’ and r,(COO-) at 526 cm-‘, are also modified as well as the 6,(NH,) (15 13 cm-‘) and y,(NH2) (1128 cm-‘) bands of the amino group. All this seems to indicate that the carboxylic and amino groups are involved in the coordination with Cr(II1) [ 121. With respect to the bands corresponding to vibrations of the hydroxyl group, most of them overlap with nucleotide bands and no information can be inferred from these data. Although some of the v(ring) bands show slight modifications, the most noticeable changes are observed in the phosphate group vibrations, especially in the symmetric stretching PO3 vibration which is shifted to higher frequencies suggesting interaction with chromium [2 11. The infrared spectra of glycine ternary derivatives show important changes for the COO- vibrations bands which seem to indicate direct interaction between the chromium and this group. The 1416 cm-’ (v,(COO-)) and 507 cm-’ (7,(COO-)) bands shift in both complexes to lower and higher frequencies respectively, whereas other carboxylic bands show slight modifications. The bands related to the amino group vibrations overlap in some cases with nucleotide bands and are not observable. With relation to the phosphate bands, the noticeable shift at higher frequencies (997 cm-‘) for the symmetric stretching band suggests coordination through this group [18, 191. The IR data for the S’AMP derivative show slight variations in the bands corresponding to tiring), but no clear conclusion can be drawn from these data. In the case of Cr&ly)2(5’CMP)(OH),* 7Hz0, the bands assignable to v(ring) (1531, 1499 and 1407 cm-‘) shift to lower frequencies and the band at 1368 cm-’ is not observed. These changes are similar to that of Co(en)2(5’CMP)(5’CMPH)*6Hz0 of which the 13C NMR spectrum suggested coordination through N(3) [22]. For this reason, participation of the cytosine ring in bonding may not be ruled out. The diffuse reflectance spectra are collected in Table 3. These data agree with a pseudooctahedral geometry for Cr(II1) bonded to N and 0 donors. The vr and v2 bands appear with several peaks which may be ascribed to splitting of 4Tzs and 4Trg(F) terms owing to distortions from Oh symmetry. For the S’CMP derivatives the changes in the ring bands rr -+ rr* and n -+ rr* [23] agree with participation of cytosine ring in coordination to Cr(II1). Except for the serine derivative, the other 5’AMP complexes show only very small changes on the UV ring bands, which suggests no direct interaction between Cr(II1) and the adenine ring. In the case of Crz(L-ser)(5’AMP)2Cl* 16Hz0, the UV bands at 252 and 293 nm in the nucleotide shift to a higher wavelength, which implies an electronic charge redistribution owing to the participation of the adenine ring in the bonding to Cr(II1).
139
In an attempt to provide further information about the ligand field environments of Cr(III) in the ternary complexes we have studied their EPR spectra. However, all these complexes, except Cr*(L-ser)(5’AMP)2C1*16Hz0, had only a broad band in the geff = 2 region, which suggests a polynuclear structure, with consequent dipolar broadening. The compound Crz(L-ser)(5’AMP)2C1* 16HsO gave a spectrum with several overlapping bands in the range 0.150.4 T, a result which suggests distortion from octahedral geometry, as indicated by the electronic spectrum.
Acknowledgements This work has received financial support from the DGICYT, project number PB86-0074-02. We are grateful to the Government of Spain for the award of a Pre-doctoral Fellowship (to A.M.C.). We also thank the Government of Pakistan for a Scholarship and the University of Baluchistan for leave of absence (to I.H.).
References 1 1. Barrett, P. O’Brien and J. Pedrosa de Jesus, Polyhedron, 4 (1985) 1. 2 J. A. Copper, L. F. Blackwell and P. D. Buckley, Inorg Chim. Acta, 92 (1984) 23. 3 E. W. Toepfcr, W. Mertz, M. M. Polansky, E. E. Roginski and W. R. Wolf, J. Agric. Food Chem., 25 (1977) 162.
4 M. Vicens, J. J. Fiol, A. Tcrron, V. Moreno and D. M. L. Goodgame, Inorg. Chim. Acta, I57 (1989) 127. 5 M. Vicens, M. Prats, J. J. Fiol, A. Terron and V. Moreno, Inorg. Chim. Acta, 1.58 (1989) 59. 6 D. Dunaw-ay-Mariano and W. W. Clcland, Biochemistry, 19 (1980) 1506. 7 S. Okada, N. Suzuki and H. Ohba, J. Inorg. Biochem., 19 (1983) 95. 8 F. Dee Snell, Encyclopedia of Industrial Analysis, Vol. 17, Wiley, New York, 1973, p. 67. 9 G. Braucr, Quimica Inorganica Preparativa, Revert& Barcelona, 1958, p. 815. 10 G. R. Brubaker, M. K. Yoo and J. R. Thuot, Gov. Rep. Announce Index, 78 (1978) 114. 11 C. A. McAuliffc, J. V. Quagliano and L. M. Vallarino, Inorg. Chem., 5 (1966) 1996. 12 K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds, Wiley, New York, 4th edn., 1978. 13 V. I. Spitsyn, S. V. Mozgin, M. G. Felin, N. A. Subbotina and A. I. Zhirov. Russ. J. Inorw. Chem.. 27 (1982) 386. 14 B. Bleaney and’K. W. H. Stevens, Rep. Prog. Whys., 16 (1953) 108. 15 E. Pedersen and H. Toftlund, Inorg. Gem., 13 (1974) 1603. 16 E. Pedersen and H. Toftlund, Inorg. C/tern., 14 (1975) as. 17 W. T. M. Andriessen and M. T. Groenewege, Inorg. Chem., I5 (1976) 621. 18 M. Tsuboi, in Basic Principles in Nucleic Acid Chemistry, Vol. 1, P.O.P. TS’O, Academic Press, New York, 1974. 19 K. A. Hartman, R. C. Lord and G. J. Thomas, Jr., in J. Duchesne (ed.), Physicochemical Properties of Nucleic Acids, Vol. 2, Academic Press, London/New York, 1973. 20 H. A. Tajmir-Riahi and T. Theophanides, Inorg. Chim. Acta, 80 (1983) 183. 21 K. Maskos, Acta Biochimica Polonica, 26 (1979) 249. 22 J. J. Fiol, A. Terron, D. Mulet and V. Moreno, Inorg. Chim. Acta, I35 (1987) 197. 23 L. B. Clark and I. Tinoco, Jr.. I. Am. Chem. Sot., 87 (1965) 11.
(1990) 133-139
133
Ternary Chromium(III)-Nucleotide-Amino L-Serine and Glycine Derivatives
Acid Complexes:
L-Methionine,
A. M. CALAFAT, J. J. FIOL, A. TERRON Departament de Quimica, Universitat de les Illes Balears, 07071 Palma de Mallorca (Spain)
V. MORENO Facultad de Quimica, Universitat de Barcelona, 43005
Tarragona (Spain)
D. M. L. GOODGAMEand I. HUSSAIN Department
of Chemistry, Imperial College, London SW72AY
(U.K.)
(Received May 22,1989; revised September 22,1989)
Abstract The first ternary Cr(III)-nucleotide-amino acid complexes (nucleotide: S’AMP, 5’CMP; amino acid: L-serine, L-methionine, glydne) are described. The complexes have been characterized by elemental and thermogravimetric analyses, IR and electronic spectroscopy and EPR measurements. In all cases the interaction of Cr(II1) with the nucleotide seems to occur mainly through the phosphate group, whereas the amino acid binds to Cr(II1) through the carboxylic and amino groups. Some of the complexes show distortions from octahedral geometry, but these distortions appear to be small resulting in values of the zero field splitting parameter D < 0.1 cm-‘. Introduction Although the role of chromium in biological systems is not perfectly known, there is abundant evidence that Cr(II1) is present in the glucose tolerance factor (GTF) [I, 21. For this reason, interest in derivatives of Cr(II1) with amino acids has increased during recent years [2-51. There is also great interest in obtaining inert derivatives of Cr(II1) with nucleotides, which could be used as enzymatic labels of allosteric enzymes [6] and for the study of the role of chromium in transcription processes and RNA and DNA interactions [7]. In this paper we describe the syntheses and characterization of the first ternary complexes of Cr(II1) with L-methionine, L-serine and glycine and the nucleotides 5’AMP and 5’CMP. Experimental Carbon, hydrogen, nitrogen and sulphur analyses were carried out with a Carlo Erba model 1106 0020-l 693/90/$3
SO
microanalyzer at the Centro de Investigation y Desarrollo-C.S.I.C. in Barcelona. Chlorine was determined by the Schoniger method. Chromium was determined by atomic absorption with a Perkin-Elmer 703 spectrophotometer using [Cr(urea)6]C1s*3Hz0 solutions as a standard. The phosphorus content was determined using the calorimetric method with phosphomolybdovanadate [8]. Thermogravimetric analyses were carried out with a Perkin-Elmer TGS-2 system with oxygen atmosphere at a velocity rate of 5 or 10 “/min depending on the cases. Conductivities were measured with a Crison 525 conductimeter at 25 “C in lop3 M aqueous solution. The IR spectra were recorded in solid state (KBr pellets) on a PerkinElmer 683 IR spectrophotometer connected to a Perkin-Elmer 3600 data station. The reflectance spectra were obtained with a Perkin-Elmer W-Vis spectrophotometer with an integrating sphere attachment. UV-Vis spectra were recorded in the same The EPR apparatus at 10e3- 1O-4 M concentration. spectra were measured using polycrystalline samples at room temperature. X-band spectra were obtained with a Varian E 12 spectrometer and Q-band spectra with a Bruker ER 200 D-SRC spectrometer and an ER 078 15-inch electromagnet (Imperial College, London). The nucleotides and amino acids (Fig. 1) were from Serva and Merck and used without further purification. The starting complex [Cr(urea)e] C13. 3Hz0 was prepared according to the literature [9]. Preparation Cr(_wnet)(L-metH)C12*3H20, Cr(L-ser)2(H20)2Cl. Hz0 and Cr(glyH)2(gly)C12*3H20 A 10 ml water solution containing 1 mmol of [Cr(urea)6]C13*3Hz0 (pH adjusted to 6.3) was added to a solution of amino acid (2 mmol in the case of L-methionine and L-serine and 3 mmol for glycine) 0 Elsevier Sequoia/Printed
in Switzerland
134
H,N-GH,-hOOH
(Gly)
GLYC INE
NH2
L-SERINE
HO-CH2-CH-COOH 2 1 3
( L-Ser)
L-METHIONINE(L-Met)
H3~-S-~H2-~H2~~H~~OOH
AH2 1;I”2
NH2
OH OH
OH OH 5’ AMP
5’ CMP
Fig. 1. Nucleotide used.
and amino
acid formulae
precipitate was formed. It was filtered off, washed with cold water and vacuum dried over P40i0. Cr2(L-ser)(5’AMPJ2CI.16H20 and Cr2(L-ser),(S’CMP)OHCl* 7H20 To an aqueous solution of Cr(L-ser)z(Hz0)2C1. Hz0 (0.94 mmol in 10 ml) was added dropwise a solution of Na25’XMP (0.94 mmol in 10 ml water, pH = 7). The violet resultant solution (pH = 4.54.7) was placed in a thermostated bath at 50 “C with constant stirring for 20 h (for the S’AMP complex) or 8 h (in the case of the S’CMP derivative). The final violet solution (pH = 3.4-3.6) was concentrated to 5 ml and then eluted through a Sephadex G-10 column to give two fractions: the first grey-blue, and the second violet. Addition of ethanol to the blue fraction and further evaporation gave and Cr*(L-ser),Cr’L(L-ser)(5’AMP),C1* 16Hz0 (S’CMP)(OH)Cl~7H,O, which are insoluble in water. These precipitates were vacuum dried over P40,e.
and abbreviations
in 10 ml water [pH adjusted to 9.2 (met and ser) and 9.8 (gly)]. The resultant green solution [pH = 9.2 (met), 9.3 (ser) and 9.7 (gly)] was placed in a thermostated bath at 50 “C, with constant stirring, for 10 h (5 h in the case of methionine complex), by which time a lilac-violet solution [pH = 3.7 (met), 3.3 (ser) and 3.9 (gly)] was obtained. This was concentrated in a rota-vapor and then eluted through a Sephadex G-10 column to give two fractions: the first one, violet, corresponding to the binary complex, and the second one, green, corresponding to unreacted starting complex. In the case of the glycine complex, there were three fractions: the first one, blue, in a very small quantity; the second, violet, corresponding to the binary complex; the third, green, corresponding to unreacted starting complex. A precipitate corresponding to the violet fraction was obtained on evaporating the solution or adding ethanol (for the L-serine complex). This was vacuum dried over P,O,,,. The three complexes are hygroscopic and soluble in water. Cr(L-ser)2(L-serH)Cl*3H20 This was obtained by the same procedure as Cr(L-ser)2(H20)2C1*H~0, but using a 1:3 Cr:L-ser stoichiometry. Cr(L-met)(5’AMP)*$H20 and Crz(L-met)(5’CMP)20H~10Hz0 Ten ml of an aqueous solution (pH approx. 7) containing 0.75 mmol of Na25’XMP were added dropwise to a solution of Cr(L-met)(L-metH)&. 3HaO (0.75 mmol in 10 ml HzO, pH 3.9-4.2). The violet mixture (pH 5.6-5.7) was maintained at 50 “C, with constant stirring, for 1.5-2.5 h. A grey
Cr2(gly)2(5’AMP)(OH)2*6H20 and Cr2(gly)2(5’CMP)(OH),~6H,0 Ten ml of an aqueous solution containing 0.64 mmol Na25’XMP (pH adjusted to 7.0) was added dropwise to an aqueous solution of Cr(gly)(glyH),C12*3Hz0 (0.64 mmol in 10 ml). The violet solution (pH 5.5 or 6.1) was maintained at 50 “C with continuous stirring for 5 or 8 h, depending on the nucleotide. By this time a precipitate was formed. This was filtered off, washed with cold water and then vacuum dried over P40io. The composition of the complexes and the analytical results are reported in Table 1. The elemental analyses were confirmed by the thermogravimetric studies, which are summarized in Table 2.
Results and Discussion The Cr(III)-amino acid binary complexes are very hygroscopic and soluble in water. Their molar conductivities are consistent with non-coordination of the chlorine atoms to chromium [lo]. The fact that the molar conductivity value of Cr(L-met)(L-metH)C12*3H20 is greater than expected for an electrolyte 1:2 may be explained as a result of dissociation equilibrium from the amino acid. The infrared spectra of these complexes show in all cases, modifications of the bands related to vibrations of the carboxylic group suggesting that this group is involved in the coordination to Cr(III) [ II131. The most important changes occur in the v,(COO-) bands (1412, 1420 and 1416 cm-i for L-met, L-ser and gly respectively) which are shifted 20-30 cm-’ to lower frequencies. There are also noticeable modifications in the (500-600) cm’
135
0
N
136 TABLE
2. Thermogravimetric
Compound
Cr(L-met)(L-metH)Cla*3HaO
Cr(L-met)(S’AMP)*11/2HaO
data for the complexes
Temperature
(“C)
30-l 30 130-980 residue 30-120
Tentative
Weight loss (%)
assignment
Calc.
Found
3.19 85.46
3.82 86.65
9.78
10.28
;HaO
68.31
2HaO + met + ado chromium phosphate
(esp.)
(esp.)
Hz0 2HaO + 2Cl+ 2met chromium oxide
120-980 residue
70.21
30-l 10 110-980 residue
8.24 66.43
8.33 66.54
5HaO met + 2cyd + 5HaO chromium phosphate
Cra(L-serH)a(L-ser)4C12.6H20
30-l 10 110-380 380-670 residue
5.94 73.28 9.18
6.25 73.26 9.10
3HaO 6ser + 2HaO Ha0 + 2Cl chromium oxide
Cr(L-ser)a(HaO)aCl*HaO
30-90 go-550 residue
5.15 80.55
5.21 79.23
Ha0 2H20 + 2ser + Cl chromium oxide
30-l 20 120-980 residue
6.65 69.05
6.67 71.13
30-l 10 110-980 residue
7.36 71.44
1.29 71.72
5HaO 1 lHa0 + Cl + 2ser + ado chromium phosphate (esp.)
30-600 residue
87.27
85.57
31~20 + 3gly + 2Cl chromium oxide
30-l 10 110-980 residue
9.74 61.32
9.12 60.80
4H20 2HaO + 2gly + ado chromium phosphate
(esp.)
30-l 20 120-980 residue
7.36 63.56
3HaO 4HaO + 2gly + cyd chromium phosphate
(esp.)
Cra(L-ser)(S’AMP)aCI.l6HaO
bands due to S(COO-), r,(COO-) and r,(COO-). The modifications of the 6,(NH2) bands at 15051514 cm-’ may be due to the interaction with Cr(II1) or to the participation of this group in hydrogen bonding [12]. The small variations in the bands arising from vibrations n(OH) at 805 cm-’ and V(S-CHs) at 13 19 cm-’ do not necessarily imply coordination of these groups to Cr(III). For Cr(L-met)(L-metH)C1,.3H,O, the bands at 578 and 450 cm-’ have been tentatively assigned to v(Cr-0) and y(Cr-N) respectively. There is no evidence of the presence of y(Cr-Cl) bands, in agreement with the conductivity measurements. Table 3 records the electronic spectral data which are in agreement with coordination of chromium to N and 0 donors in a pseudooctahedral geometry. These results are confirmed by EPR measurements. The X-band EPR spectra of the complexes Cr(L-met)(L-metH)Clz*3Hz0 and Cr(L-ser),(H,O),Cl.H,O,
4H20 + 2ser + cyd + Cl chromium phosphate (esp.)
7.22 62.76
gave a broad featureless band in the g,, = 2 region, characteristic of Cr(II1) in a pseudooctahedral environment. However, Cr(glyH)2(gly)Clz*3Hz0 and Cr(L-ser),(L-serH)Cl*3Hz0 gave EPR spectra comprising several overlapping bands in the range 0.150.4 T (Fig. 2). Such spectra may be described by the spin Hamiltonian for S = $ systems of the form ;fc = Pk.&S,
+&J&S,
+gJL%)
+ D(S,2 - 5/4) t E&2
- S,‘)
in which the second and third terms are the axial and rhombic zero field splittings (zfs) respectively [ 141. Although the individual band components are insufficiently resolved for accurate evaluation of D and E parameters, the facts that the strongest transition appears in the geff = 2 region, the bands below 0.2 T are very weak, and no bands are observed between 0.5-l T indicate a relatively low value for
137 TABLE
3. Electronic
spectral
data for the complexes
Compound
(bands
in nm)*
A-n* n-+n*
W4T
r,(P)
- 4A2a
(v J4Tza
+ 4A*g
10 Dq (cm-‘)
Cr(L-met)(L;metH)Cla.3H20
404(89)
548(41)
18200
Cr(L-ser)a(H20)2C1.H20
403(110)
541(60)
18500
Cra(L-serH)a(L-ser)4Cla.6H20
401(152)
541(105)
18500
403(84)
544(51)
18400
435s 395s
610s 570s 470sh
18200
440s 395s
610s 565s 500sh 465sh
18700
435s 395s
610s 570s 470sh
18200
27Os, 310s
435s 395s
610s 570s
17000
280sh,
435s 395s 365sh
615s 560s SOOsh 465sh
18700
435s 400s
610s 575s 500sh 470sh
18600
Cr(glyH)a(gly)C12.3Ha0 Naa5’AMP
b
252s, 293s
Cr(L-met)(5’AMP).11/2H20
b
Cra(L-ser)(S’AMP)2C1.16H20
b
Cr&ly)a(5’AMP)(OH)a.6H20
Naa5’CMP
255s
% = strong,
270br,
b
m = medium,
325s
255s, 290s
b
26Os, 308s
Cra(L-met)(5’CMP)2(0H).10H20
Cra(gly)a(5’CMP)(OH)2.7H20
295s
b
b
br = broad,
270s
325s
310s
sh = shoulder,
sp = sharp, w = weak.
D (
bDiffuse
reflectance
spectra.
ligand are not observed. However, this fact does not necessarily imply coordination through sulphur atom. In both complexes, the v,(COO-) band is shifted by 20-30 cm-’ to lower frequencies. The bands assignable to amino group vibrations are modified in the spectra of the complexes or overlap with nucleotide bands. With regard to nucleotide vibrations, there are small variations in some Y(ring) bands (1600-1200 cm-‘). In the case of the S’CMP compound, a new band appears at 1720 cm-’ which may be due to a free carboxylic group or to participation of C=O in hydrogen bonding upon complexation [18, 191. Nevertheless, the most important changes occur in the phosphate bands, especially that of u,(POa) band (978 cm-‘), which is shifted by 20-25 cm-’ to higher frequencies in both cases. Therefore, IR data suggest that methionine interacts with chromium through its carboxylic and amino groups [ll, 121 and the phosphate group of S’AMP and S’CMP is involved in metal coordination [ 18,201. In the case of serine ternary compounds again there are important changes in the bands correspond-
138
2
1
L
3
5
6 10’
mT
Fig. 2. X-band EPR spectra of polycrystalline samples of: (a) Cr+ser)(S’AMP)?Cl.l6HzO; (b) Crz(L-serH)2(L-ser)4C12*6Hz0; (c) Cr(glyH)2(gly)Clz*3HzO.
/
g=
(b)
.‘M,
9:
Cc)
C.-
/.‘. .-
.f ,
/:
1:
i! ;:
/// 9=
‘
6
.3
IO
12
102.7
Fig. 3. Q-band EPR spectra of pofycrystalline samples of: (a) (-. -) Crz(L-ser)(S’AMP)$l*16H20; (b) (-) CIZ(LserH)&ser)&12*6HzO; (c) (- - - -) Cr(glylI)~(gly)Cl~.3H~O.
ing to carboxylic vibrations. The band assignable to v,(COO-) shifts 25-30 cm-’ to lower frequencies, overlapping with -/,(CH,) + v(C-0) of the serine
(1384 cm-‘). The carboxylic bands at lower frequency, S(COO-) at 612 cm-’ and r,(COO-) at 526 cm-‘, are also modified as well as the 6,(NH,) (15 13 cm-‘) and y,(NH2) (1128 cm-‘) bands of the amino group. All this seems to indicate that the carboxylic and amino groups are involved in the coordination with Cr(II1) [ 121. With respect to the bands corresponding to vibrations of the hydroxyl group, most of them overlap with nucleotide bands and no information can be inferred from these data. Although some of the v(ring) bands show slight modifications, the most noticeable changes are observed in the phosphate group vibrations, especially in the symmetric stretching PO3 vibration which is shifted to higher frequencies suggesting interaction with chromium [2 11. The infrared spectra of glycine ternary derivatives show important changes for the COO- vibrations bands which seem to indicate direct interaction between the chromium and this group. The 1416 cm-’ (v,(COO-)) and 507 cm-’ (7,(COO-)) bands shift in both complexes to lower and higher frequencies respectively, whereas other carboxylic bands show slight modifications. The bands related to the amino group vibrations overlap in some cases with nucleotide bands and are not observable. With relation to the phosphate bands, the noticeable shift at higher frequencies (997 cm-‘) for the symmetric stretching band suggests coordination through this group [18, 191. The IR data for the S’AMP derivative show slight variations in the bands corresponding to tiring), but no clear conclusion can be drawn from these data. In the case of Cr&ly)2(5’CMP)(OH),* 7Hz0, the bands assignable to v(ring) (1531, 1499 and 1407 cm-‘) shift to lower frequencies and the band at 1368 cm-’ is not observed. These changes are similar to that of Co(en)2(5’CMP)(5’CMPH)*6Hz0 of which the 13C NMR spectrum suggested coordination through N(3) [22]. For this reason, participation of the cytosine ring in bonding may not be ruled out. The diffuse reflectance spectra are collected in Table 3. These data agree with a pseudooctahedral geometry for Cr(II1) bonded to N and 0 donors. The vr and v2 bands appear with several peaks which may be ascribed to splitting of 4Tzs and 4Trg(F) terms owing to distortions from Oh symmetry. For the S’CMP derivatives the changes in the ring bands rr -+ rr* and n -+ rr* [23] agree with participation of cytosine ring in coordination to Cr(II1). Except for the serine derivative, the other 5’AMP complexes show only very small changes on the UV ring bands, which suggests no direct interaction between Cr(II1) and the adenine ring. In the case of Crz(L-ser)(5’AMP)2Cl* 16Hz0, the UV bands at 252 and 293 nm in the nucleotide shift to a higher wavelength, which implies an electronic charge redistribution owing to the participation of the adenine ring in the bonding to Cr(II1).
139
In an attempt to provide further information about the ligand field environments of Cr(III) in the ternary complexes we have studied their EPR spectra. However, all these complexes, except Cr*(L-ser)(5’AMP)2C1*16Hz0, had only a broad band in the geff = 2 region, which suggests a polynuclear structure, with consequent dipolar broadening. The compound Crz(L-ser)(5’AMP)2C1* 16HsO gave a spectrum with several overlapping bands in the range 0.150.4 T, a result which suggests distortion from octahedral geometry, as indicated by the electronic spectrum.
Acknowledgements This work has received financial support from the DGICYT, project number PB86-0074-02. We are grateful to the Government of Spain for the award of a Pre-doctoral Fellowship (to A.M.C.). We also thank the Government of Pakistan for a Scholarship and the University of Baluchistan for leave of absence (to I.H.).
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